One of the great aspirations of gene therapy is to develop technology that will provide a feasible approach to correct genetic defects and combat infectious diseases. We are engaged in studying the molecular biology of the human parvovirus adeno-associated virus (AAV) with the intent to develop a safe and efficient viral vector for human gene therapy. AAV is a dependent parvovirus which requires co-infection with another virus [either adenovirus (Ad) or certain members of the herpes virus group] to undergo a productive infection in cultured cells. In addition to its unique life-cycle, AAV has a broad host range for infectivity (human, mouse, monkey, dog, etc.), it is ubiquitous in humans, and is completely nonpathogenic. Our research pioneered the use of recombinant AAV (rAAV) as a gene delivery system for central nervous system, and muscle cells, with vector expression for over 1.5 years without immune consequences or vector toxicity. We initiated studies that uncovered rate limiting steps involved in vector transduction. This has resulted in a new approach for generating Ad free rAAV preps. Our continued efforts to dissect the primary steps involved in AAV infection has recently led to the identification of the AAV 2 receptor. These advances and the development of novel AAV serotypes have provided a tool box like approach to engineer airway specific AAV vectors. Identification of the AAV receptor, rate limiting steps for vector transduction and ability to efficiently transduce primary brain and muscle cells, but not airway cells in vivo, has provided a unique paradigm for studying the molecular steps involved in efficient vector transduction. The overall objective of the proposed work is to study the primary steps involved in rAAV transduction and persistence in airway cells using chimeric AAV capsid evolved to specifically infect human airway epithelial cells. The long range objective is to better understand these molecular steps in primary airway cells with the ultimate goal of developing specific viral vectors with efficient targeted transducing capability of CFTR.